Phytomedicinal properties of Hygrophila schulli (Neeramulliya)

Hygrophila schulli which is known as “Neermulli’’ in the vernacular is an herbaceous plant native to Sri Lanka. Ancient medicinal literature suggests the use of H. schulli whole plant or its parts for the treatment of different communicable and non-communicable diseases including diabetes mellitus and tuberculosis. Active constituents and secondary metabolites including alkaloids, tannins, steroids, proteins, flavonoids, and glycosides are identified to possess antimicrobial, antitumor, antioxidant, hepatoprotective, anthelmintic, nephroprotective, antidiabetic, anticataract, anti-inflammatory, anti-nociceptive, hematopoietic, diuretic, antiurolithiatic, antipyretic, neuroprotection, and anti-endotoxin activities. In this review, we reviewed clinical studies, patents, and analytical studies from the earliest found examples from 1886 to the end of 2021. We critically analyzed and attempt to summarize the information based on bioactivities and chemical composition of H. schulli plant extracts which will be of future use for researchers in this field.


Introduction
Natural medicines are considered the primary sources of medicinal agents over centuries in various cultures with social norms, ethical values, and traditional customs. Herbal medicine is the pioneering branch of natural medicine because over 90% of natural medicines are plant related. Therefore, the contribution of herbal medicine to the current pharmacopeia is significant. Approximately 80% of the global populace relies on traditional herbal medicine for primary health care (1) due to various reasons including its safety, long pharmacopeial history, availability and affordability, high efficacy, and low side effects. Herbal medicines are known to have a wide range of biological activities including anti-oxidant, anti-inflammatory, anticancer, and antifibrotic activities, etc., and are widely used as alternative or complementary medicines for the prevention and treatment of diverse disease conditions (2).
Sri Lanka is considered a biodiversity hotspot with a high degree of endemism. Therefore, plants growing on this tiny island have unique features including morphological and biological features. The use of herbs as medicinal agents in Sri Lanka has been enriched by several medicinal systems including traditional folk medicine, Ayurveda, Unani, and Siddha. The use of herbal medicine in traditional and Ayurveda medicinal systems has over 3000 years of documented history.
Hygrophila schulli is one such plant, indigenous to Sri Lanka (3), which is commonly used in both traditional and ayurveda systems to treat many disease conditions. In Sri Lanka, it is known as "Neeramulliya" or "Niramulli". In this review, the various scientifically proven photochemical properties of H. schulli are discussed to strengthen the use of this valuable plant as herbal medicine. The plant's common habitat is moist or wet places, especially in Asia and African regions (4).
In Ayurveda, H. schulli leaves and young stems are frequently used for the treatment of dysentery, edema, inflammation, cough, joint pains, bacterial infections, rheumatism, renal calculi, and other renal diseases, hepatic diseases, and microbial infections such as gonorrhea and urinary tract infections, etc. (12). It has been found that H. schulli mainly contains alkaloids, lupeol, stigmasterol, isoflavone, glycoside and uncharacterized bases, etc. (13). However, the chemical constituents of the plant extracts are varying depending on the geographic origin of the plant, the solvents to be used for extractions and conditions to be applied, etc. (5). Therefore, contradictory data have been reported regarding the phytochemical composition of the different extracts of H. schulli. This review analyses the available scientific evidence on different bioactivities of H. schulli extracts as an alternative therapeutic strategy for various lifestyle diseases that require lifelong pharmaceutical medication to raise the quality of individuals' lives.
H. schulli plant is an annual, approximately 1.5 m in height spiny herb (12). In general, it contains eight leaves and six spines at each node. The leaves are sessile, whorled, and have undulating margins (5). Flowers arise from October to December and are 2-3 cm long, purple-blue with a 4-lobed calyx (11,14). There are about 4-8 orbicular seeds on the hard retinacula which are 7.5 mm in length and 0.3 mm across with a linear-oblong capsule (14). According to the International Union for Conservation of Nature (IUCN) 2011, H. schulli species has been identified under the least concern group (16) (Figure 1).

Bioactivities of Hygrophila schulli Antimicrobial Activity of H. schulli
Many studies have been conducted to determine the antibacterial and antifungal activities of H. schulli extracts. We have noted that the antimicrobial activity of a given extract depends on various factors such as extraction method, solvents used, parts of the plant used to extract phytochemicals, and especially, the mode of antimicrobial action and the effective concentrations depending on the constituents present in the extract and their abundance.
The multidrug resistance (MDR) of pathogenic microbial species is becoming an emerging problem in the healthcare setting. The emergence of MDR is leading to the identification of novel therapeutic alternatives, also studies on their clinical applicability, toxicity, and mode of action are becoming a necessity when the health effects of MDR are concerned (17). In this case, plant derivatives act as a potential drug candidate with high potency, low toxicity, wide availability, and accessibility (18). Importantly, pathogens usually do not develop MDR against plant-based antimicrobials. Furthermore, many plant species are rich in secondary metabolites with antimicrobial activity. Thus, natural herbal products based on novel antimicrobial drug discovery are exhibiting great success (19,20). Table 1 summarizes the findings of some studies done to detect the antimicrobial activity of H. schulli plant extract.

Anti-biofilm Activity of H. schulli
Even though many studies show the antimicrobial activity of H. schulli extracts, their anti-biofilm activity is not well studied yet. Biofilm is a complex arrangement of different microorganisms. Microbial biofilms are cellular aggregates covered with exopolymeric substances (EPS) attached to both biotic surfaces such as host cells and abiotic surfaces such as medical devices. Microbial biofilms are inherently resistant to routine antimicrobial agents compared to planktonic counterparts, due to their molecular contents such as eDNA and exoenzymes, reduced diffusion of antimicrobial agents through the biofilm matrix, persistent cellular content, and limited nutrients and oxygen (29). Global statistics reveal that the majority of chronic human infections are biofilm related. Therefore, it is very important to investigate effective antimicrobial agents with antibiofilm activity. In this case, phytotherapeutic approaches become a potential candidate. Therefore, future studies were recommended to identify plant species with anti-biofilm effects.

Anti-oxidant and free radical scavenging activities of H. schulli
Anti-oxidants are emerging as therapeutic and prophylactic agents which scavenge free radicals like Reactive Oxygen Species (ROS) and reduce the damage caused by them (30). Free radicals have the potential to destroy healthy cells of the body by damaging their structure and functions (5). Free radicals are accountable for causing a tremendous number of diseases and health conditions which include cardiovascular diseases, cancers, Alzheimer's disease, neural disorders, mild cognitive impairments, alcohol-induced liver disease, Parkinson's disease, ulcerative colitis, atherosclerosis, and aging (5,31). Oxidative stress which is an imbalance between oxidants and anti-oxidants leads to many biochemical changes. Recently, interest in searching for naturally occurring antioxidants has increased considerably due to the adverse side effects of synthetic anti-oxidants, such as hepatotoxicity, carcinogenicity, and nephrotoxicity (32).
The medicinal plant, H. schulli is commonly prescribed by ayurvedic practitioners as an anti-oxidant and free  Table 2.

Hepatoprotective activity of H. schulli
The liver is a vital organ of the human body that plays a pivotal role in regulating various physiological activities in the body. It regulates numerous vital functions, such as metabolism, storage, and secretion. It has a huge capacity to detoxicate toxic substances such as caffeine and alcohol and synthesize useful substances such as blood clotting factors. Therefore, damage to the liver by hepatotoxic agents is of huge consequence (33). Still, there are no effective drugs in modern medicine, to regenerate hepatic cells and stimulate liver functions (34). In traditional and ayurvedic medicine, many medicinal plant preparations are recommended for the treatment of liver disorders and often offer significant relief (35). Scientific evidence reveals that the extractions of H. schulli achieve great success in the treatment of hepatic disorders ( Table 2).

Anti-diabetic activity of H. schulli
There has been an exponential increase in the occurrence of diabetes in modern society, due to the recent lifestyle deviations and shift towards excessive urbanization. Globally more than 420 million people are estimated to be suffering from diabetes mellitus. Indigenous plant-based treatments are greatly useful due to the high cost and possible toxicity associated with Western medicine. However, crude drugs derived from plants must also be subjected to extensive phytochemical analysis to ensure efficacy and safety and to develop sustainable, safe, and marketable therapeutic drugs (36). With the high occurrence of diabetics in society, scientists pay attention to finding novel ways to treat and control diabetes and associated complications, especially with the use of phytomedicinal components.
Due to extensive folk medicine systems like Ayurveda and Siddha, Sri Lanka is blessed with a wealth of ancestral knowledge about medicinal plants like H. schulli. Extracts of H. schulli hold greater promise for a country like Sri Lanka where there is an unprecedented rise in patients suffering from diabetes mellitus. Table 2 summarizes the findings of in vitro and in vivo studies on the anti-diabetic activity of H. schulli plant extracts.

Anti-nociceptive and anti-inflammatory activities of H. schulli
The management of pain and inflammation with routine opioid analgesics and non-steroidal anti-inflammatory drugs is recently encountering severe adverse effects like tolerance, dependence, and gastrointestinal problems (4,37). Currently, traditional medicinal plants are widely used as alternative remedies for the treatment of pain and inflammation. H. schulli is one of the traditionally used medicinal plants for the treatment of pain and inflammatory conditions (11). Moreover, most of the studies show that the seed extract of H. schulli possessed significant antinociceptive and anti-inflammatory activities ( Table 2). H. schulli leaves macerated in alcohol are used for the management of headaches while fresh leaves of H. schulli are used against inflammatory conditions in the skin (4).
Due to the adverse effects arising with the use of common anti-cancer drugs, traditional medicinal plants have been investigated as an alternative source of therapeutic agents which potentially had lesser adverse side effects. The use of natural medicinal plants as therapeutic agents is becoming more popular in the modern world. Toxicological assessment of a natural product is an important aspect that should be concerned before the clinical application (19). Modern scientists conduct studies on the cytotoxic effects of H. schulli to ensure the safe use of this important phytochemical agent.
Several toxicological studies have proved that at a dose of 2000 mg/kg body weight (BW), H. schulli plant extracts had not expressed any significant changes in the biochemistry parameters, weight of the internal organs, body weight, and food and water consumption of experimental animal models (53). Although H. schulli plant extracts were found to have no cytotoxic effect against normal cells, they produced selective cytotoxic activity towards tumor cells at a range of IC50 0.22-1.6 mg/ml (1,13). Importantly, some controversial findings were reported by various scientists on the toxicological characteristics of H. schulli extracts. The extraction method, solvents used, geography and botanical properties of the plant, and environmental factors can influence such discrepancies. Table 3 summarizes the findings of in vitro and in vivo studies on anti-cancer, anti-tumor activities, and cytotoxicity of H. schulli plant extracts.

Chemical composition of Hygrophila schulli
Phytochemicals obtained from medicinal plants play a significant role in medicinal science to cure or hinder critical health conditions. Researching phytochemical screening has created a new bridge between conventional and modern drug compounds (5). Phytochemical screening is usually carried out on the whole plant or sometimes on a particular part of the plant (stem, leaves, roots, seeds, etc.) to get the desired bioactive compounds (57).
Phytochemical constituents and their relative abundance Table 3. In vivo studies on anti-cancer, anti-tumor activities, and cytotoxicity of Hygrophila schulli plant extract play a major role in the determination of the biological activity and efficacy of phytomedicinal preparations. The chemical composition of the extract depends on various facts such as extraction method, solvents used, and parts of the plant used to extract phytochemicals. Ethnomedicinally it has been reported that the plant H. schulli is used for the treatment and prevention of various human disease conditions such as arthritis, allergies, anemia, cancers, hypertension, body pain, constipation, cough, renal dysfunction, diarrhea, dysentery, fistula, edema, gall stones, urinary calculi, kidney stone, inflammations, leprosy, jaundice, liver disorder, skin disease, leucorrhoea, rheumatism, tuberculosis, malaria, urogenital disorder, and venereal diseases.
The anticancer activity of H. schulli is due to the presence of phenols and flavonoids. Phenolic compounds reduce the formation of pre-cancerous cells. Flavonoids may protect DNA from oxidative damage, inhibit carcinogen activation, activate carcinogen detoxifying systems, and interfere with the development of malignant tumors (55,60).
Ethanolic extract of H. schulli leaf demonstrated anti-inflammatory properties. This may be due to the hyaluronidase inhibition property of the extract. Flavonoids and tannins are some such hyaluronidase inhibitory compounds present in H. schulli extracts. Hyaluronidase is an enzyme that degrades β1, 4 glycosidic linkage of hyaluronic acid which leads to the activation of proinflammatory cytokines. Whenever there is inflammation, tissue permeability is enhanced by the hyaluronidase and spread the inflammatory responses around the affected organ. Furthermore, secondary metabolites such as stigmasterol, lupeol, and lup-20 (29)-ene-3β,23-diol also contribute to the anti-inflammatory activity of H. schulli leaf extract (4).
The availability of flavonoids, sterols, terpenoids, aliphatic esters, and botulin among the plant constituents contributes to the protective effect of H. schulli against hepatotoxins. The flavonoids are known antiperoxidants, anti-oxidants, and free radical scavengers leading to hepatoprotection (3).
Phytoconstituents such as tannins, flavonoids, alkaloids, phenolics, compounds, and saponins are responsible for the antibacterial effect. Irreversible complexes are formed by tannins with proline-rich proteins, resulting in the inhibition of cell wall synthesis. Lipophilic flavonoids exhibit antimicrobial activity by reacting with lipid components and disrupting microbial cell membranes.
Seeds of H. schulli are a potential source of anti-oxidants that prevent DNA damage. The presence of flavonoids and phenolic compounds like quercetin and tricin in H. shulli seeds prevents the production of ROS by complexing cations, indicating the protection of DNA (16).

Conclusion
Based on the available literature evidence, phytoconstituents extracted from various parts of H. schulli act as reservoirs of medically important phytochemical agents. Although different parts of the H. schulli plant were found to have various medicinal properties against infections and diseases, its anti-biofilm properties are not elucidated yet. Most of the bioactivities of H. schulli are due to the presence of phytoconstituents such as flavonoids (ellagic acid, apigenin, quercetin, luteolin, and gallic acid), alkaloids (asteracanthicine and asteracanthine), triterpenes (hentricontane, lupeol, lupenone, and betulin), sterols (asterol and stigmasterol), fatty acids, minerals, aliphatic esters, amino acids, and essential oils. The potential bioactivities, mechanisms of action, chemical profiles, and toxicological assessments of various plant extracts and their fractions provide the necessary information to develop novel therapeutic alternatives with high efficacy, availability, and low toxicity using natural sources.